Glucose responsive insulin secreting β-cell lines and method for producing same

ABSTRACT

A line of β cells is described which is capable of maintaining high levels of insulin secretion in culture. Such cells are useful in the treatment of diabetes, for example, by encapsulation of the cells in an insulin-permeable membrane device, followed by implantation into a diabetes patient.

This application is a division application of U.S. patent applicationSer. No. 08/208,873, filed Mar. 10, 1994, now U.S. Pat. No. 5,534,404which is a continuation application of U.S. patent application Ser. No.08/165,088, filed Dec. 10, 1993, now abandoned.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to insulin-secreting β cells and methods ofproducing β cell populations having desirable features.

2. Description of Art

The insulin producing tissue of the pancreas, the islets of Langerhans,constitute a small fraction of the organ. Islets are largely composed ofsmall clusters of β-cells and there is a need to develop a reliablesource of β-cells which respond to glucose stimulation in a mannersimilar to that of normal islet cells for diabetes research and forimplantation into diabetic subjects.

Populations of β-cells are known to show considerable heterogeneity intheir morphology, their insulin secretion, and their glucoseresponsiveness. Normal islet tissue has been found to contain β cellswhich secrete insulin in response to glucose as well as some that donot. Individual glucose-responsive cells in the population have beenshown to secrete insulin at different glucose levels. Pipeleers,"Perspective in Diabetes Heterogeneity in Pancreatic β-Cell Population",Diabetes (1992) 41:777-780. Islet cells in primary tissue culture show acharacteristic sigmoidal curve upon glucose stimulation. As indicated inWollheim, et al., "Establishment and Culture of Insulin-Secreting β CellLines", Methods in Enzymology (1990) 192:223-235, in a native β cellfrom all but the ruminants, the half-maximum level of insulin secretionis at about 7-8 mM glucose and the maximum is at a glucose level ofabout 15 mM or so (meaning that insulin secretion is about twice as muchat 15 mM glucose as at 7-8 glucose). It is desirable, thus, that cellsdesigned to mimic the activity of normal cells, either for implantationor for testing show a similar pattern of insulin secretion.

Historically, β-cells have been obtained by isolating them from primarytissue, employing collagenase digestion of the pancreas, atime-consuming and expensive process. However, while primaryhormone-secreting cells can often be maintained for several months inculture, they generally undergo few or no cycles of cell division.During this time, the cells generally display a decrease in hormonesecretion and/or a loss of regulation. For human transplantationpurposes, researchers have investigated the use of both human and animaltissue. A major problem with the use of human tissue, however, is theshortage of available organs. Where animal tissue is used, extreme caremust be taken to obtain material from pathogen-free animals and allisolated tissue must be extensively tested. Wang, et al., "Glucose- andAcetylcholine-Induced Increase in Intracellular Free Ca²⁺ inSubpopulations of Individual Rat Pancreatic β-Cells", Endocrinology(1992) 131:146-152 and Wang, et al., "Glucose-induced Insulin Secretionfrom Purified β-Cells", The Journal of Biological Chemistry (1993)268:7785-7791 and others have sorted β cells from other pancreatictissue by fluorescence-activated cell sorting using inherentlight-scattering patterns and flavin adenine dinucleotideautofluorescence. De Krijger, et al., "Enrichment of Beta cells from thehuman fetal pancreas by fluorescence activated cell sorting with a newmonoclonal antibody", Diabetologia (1992) 35:436-443 has sorted humanislet cells from other human pancreatic tissue by producing mousemonoclonol antibodies specific to the islet cells. The antibody waslabeled and used for fluorescence-activated cell sorting, the resultingcultures showing an enriched β cell content. In general, though, thesecells do not divide, and it is costly and time-consuming to repeatedlyprepare β cells in this manner.

Some reports have indicated that β cells isolated from primary tissuecan be made to divide in vitro. For example, Brothers, in"Hormone-Secreting Cells Maintained in Long-Term Culture", PCTApplication WO 93/00441 published Jan. 7, 1993, selected and culturedcells from human pancreatic tissue without use of collagenase orcentrifugation to establish subcultures of glucose-responsive cellscultured, at least originally, in media resembling the in vivoenvironment. Subsequently, individual cells or cell clumps in culturewere selected for further propagation according to proliferation rateand amount of insulin secreted. Thawed cells which had beencryopreserved and cultured at passage 47 were tested for insulinsecretion. Insulin secretion, according to the data presented, did notshow the characteristic sigmoidal curve of correctly regulated cells,but rather what appears to be a horizontal line showing relativelystatic insulin secretion at different glucose levels. In addition,insulin levels are only at about 3.7×10³ μIU/1.5×10⁶ cells/hour.Furthermore, these cultures were not free from contaminating non-βcells.

Zayas, et al., in "Proliferated Pancreatic Endocrine Cell Product andProcess", EPO Application A2 0 363 125, published Apr. 11, 1990,discloses the culturing of pancreatic islet progenitor cells. Thesecells were proliferated in subculture in a collagen/laminate substrategel to allow a three-dimensional culture system. The undifferentiatedprogenitor cells, when implanted, are reported to differentiate in vivo,resulting in in vivo insulin secretion.

Other researchers have attempted to overcome the problems associatedwith isolating natural islet cells by developing β-cell lines. A cellline offers several advantages over the use of primary tissue, as itprovides a renewable source of cells having consistent properties.Attempts have been made to develop reliable cell lines from insulinomas.Wollheim, et al., supra, reports that a major problem with such celllines, though, is the tendency of these cells to lose theirdifferentiated status in culture, and a corresponding decrease in thecellular insulin content. As a result, most such previous approacheshave achieved only limited success. After repeated passaging in vitro,these cell lines tend to show little or no insulin secretion, and/or alack of desired insulin regulation in response to glucose.

Gazdar, et al., "Continuous, clonal, insulin- and somatostatin-secretingcell lines established from a transplantable rat islet cell tumor",Proc. Natl. Acad. Sci. USA (1980) 77:3519-3523 discloses theestablishment of cell lines of rat pancreatic islet cells devoid offibroblastoid cells by centrifuging to remove erythrocytes and enhancinggrowth by using feeder layers of rat liver cells. The final cultureswere well-isolated colonies harvested and propagated to mass cultures.However, different sublines of the cell lines show different amounts ofglucose responsiveness, and maximum insulin production shown after about100 days was about 150 to 250 μU/10⁶ cells/24 hours.

β-cell lines have been developed from X-ray induced mouse insulinomas aswell as from insulinomas in transgenic mice expressing simian virus 40 Tantigen. See Asfari, et al., "Establishment of2-Mercaptoethanol-Dependent Differentiated Insulin-Secreting CellLines", Endocrinology (1992) 130:167-178, Hanahan, "Heritable formationof pancreatic β-cell tumours in transgenic mice expressing recombinantinsulin/simian virus 40 oncogenes", Nature (1985) 315:115-122 and Efrat,et al., "Glucose Induces Insulin Gene Transcription in a MurinePancreatic β-Cell Line", The Journal of Biological Chemistry (1991)266:11141-11143. However, these cells (specifically RIN cells, HITcells, β-TC cells, and INS cells) either do not show high insulinsecretion or correct regulation and frequently do not retain theirsecretory characteristics over numerous passages. A discussion of theβ-TC-3 cell line is found in detail in Efrat, et al., supra and numerousβ-TC cell lines are discussed in D'Ambra, et al., "Regulation of InsulinSecretion from β-Cell Lines Derived from Transgenic Mice InsulinomasResembles that of Normal β-Cells", Endocrinology (1990) 126:2815-2822.Although INS cells, particularly INS-1 show some degree of regulation,they do not show a large increase between half-maximum and maximumsecretion and lower levels of secretion found in correctly regulatedcells. In addition, the INS cells are mercaptoethanol-dependent forgrowth. Asfari, supra.

Some researchers have made specific attempts to overcome various ofthese problems. Miyazaki, et al., "Establishment of a Pancreatic β CellLine That Retains Glucose-Inducible Insulin Secretion:Special Referenceto Expression of Glucose Transporter Isoforms", Endocrinology (1990)127:126-132 discloses two β-cell lines, called MIN6 and MIN7 obtained bytargeted expression of the simian virus 40 T antigen gene in transgenicmice, the former obtained using "more than one cloning step", specificteachings of these steps being absent from the article. These cells havebeen characterized at 16-23 passages by Sakurada, et al., "Relationbetween Glucose-Stimulated Insulin Secretion and Intracellular CalciumAccumulation Studied with a Superifusion System of a Glucose-ResponsivePancreatic β-Cell Line MIN6", Endocrinology (1993) 122:2659-2665 andIshihara, et al, "Pancreatic beta cell line MIN6 exhibitscharacteristics of glucose metabolism and glucose-stimulated insulinsecretion similar to those of normal islets, Diabetologia (1993)36:1139-1145. Additional information about these cells is found inHamaguchi, et al., "NIT-1, a Pancreatic β-Cell Line Established From aTransgenic NOD/Lt Mouse", The Jackson Laboratory, Bar Harbor, Me.(1991). According to Miyazaki, the MIN6 cells are regulated at 30passages, although no data is presented to characterize the quality ofregulation. It is interesting to note, as well, that while Ishihara hasalso characterized the MIN6 cells, and shown regulation at passages 16to 23, the insulin output was significantly lower than initiallyreported by Miyazaki for these same cells at passage 16, suggesting somedeterioration in insulin secretory response. However, at best thesecells are reported to secrete about 1125 μIU of insulin/45 min/10⁵cells.

Increased intracellular free Ca⁺² ("cytosolic free calcium") is known tobe induced by glucose in certain β cells. According to Wang, et al.,"Glucose- and Acetylcholine-Induced increase in Intracellular Free Ca²⁺in Subpopulations of Individual Rat Pancreatic β-Cells", Endocrinology(1992) 131:146-152, p. 149, the pattern of response in β cells issimilar to that of whole islets and isolated pancreas cells in priorstudies. Wang, et al., "Glucose-induced Insulin Secretion from Purifiedβ-Cells", The Journal of Biological Chemistry (1993) 268:7785-7791 hasshown that β cells which do not show increased calcium concentration indirect response to glucose only may do so in the presence of otheragents, resulting in increased insulin secretion in response to glucosestimulation. The presence of cytosolic free calcium in MIN6 cells (shownto be correctly regulated) and RINm5F cells (which have not shown highinsulin secretion) was investigated by Sakurada, et al., supra. A closerelationship between the rise of cytosolic free calcium concentrationand insulin secretion was reported.

Omann, et al., "Pertussis Toxin Effects on Chemoattractant-InducedResponse Heterogeneity in Human PMNs Utilizing Fluo-3 and FlowCytometry", Cytometry (1991) 12:252-259 discloses the use ofFluo-3-acetoxymethyl ester (produced by Molecular Probes, Eugene,Oreg.), hereafter sometimes referred to as "Fluo-3", which binds withCa⁺² in polymorphonuclear leukocytes for measurement of cytosoliccalcium levels induced by N-formylpeptide.

Attempts have been made to transplant both insulinoma and normal isletcells into insulin-requiring organisms. The insulinoma transplanted intorats at an extrapancreatic site by O'Hare, et al., "Influence of atransplantable insulinoma on the pancreatic status of insulin andpancreatic polypeptide in the rat", Diabetologia (1985) 15 28:157-160resulted in insulinaemia and hypoglycemia compared with con trols.Undifferentiated pancreatic islet progenitor cells were transplantedinto mice and allowed to differentiate in vivo for insulin production invivo in Zayas, et al., supra.

The implantation of islet cells is discussed generally in Lacy, "Statusof islet cell transplantation", 1 Diabetes Reviews (1993) No. 1, pp.76-92. According to Lacy, to reduce rejection of foreign cells in thehost organism, certain attempts have been made to reduce contact of theforeign cell with the host. For example, fetal rat islet cellsencapsulated in microspheres have been transplanted into mice.Biocompatibility problems encountered were reduced by coating themicrospheres with alginate. According to Lacy, mouse pancreatic cellsencapsulated in hollow fibers had prolonged survival when transplantedinto hamsters. Lacy indicates that suspending rat islets in alginate,however, while encapsulated in acrylic copolymer hollow fibers has beenshown to maintain normoglycemia in diabetic mice, using even asubcutaneous site, normally a deleterious one for islet cells. Inaddition, Hoffman, et al., in Experimental Neurology, "Transplantationof a Polymer-Encapsulated Cell Line Genetically Engineered to ReleaseNGF", (1993) 122:100-106, reports that the transplantation of ratfibroblasts or fibroblasts genetically modified to produce NGF (nervegrowth factor) were loaded within a thermoplastic hollow fiber-basedcapsule.

However, in Hicks, et al., "Transplantation of β cells from transgenicmice into nude athymic diabetic rats restores glucose regulation",Diabetes Research and Clinical Practice (1991) 14:157-164, β-cells fromthe mouse pancreatic β-cell line PTC-1, one of the cell lines mentionedabove (which does not show proper regulation and shows low insulinsecretion according to D'Ambra, supra) attached to a collagenmicrocarrier and implanted in diabetic rats show improved insulinproduction and glucose response over diabetic rats implanted only withmicrocarriers, but showed increased granuloma formation and intenseinflammatory reaction compared to diabetic controls without anyimplants.

It is thus apparent that there is still a need for the development ofdividing β-cell populations resembling normal islet cell populations ininsulin secretion levels and in correct insulin regulation in responseto glucose, particularly such cells which are phenotypically stable overtime and which can be repeatably and predictably produced, as well asimplanted for the treatment of diabetes.

The references discussed above are provided solely for their disclosureprior to the filing date of the present application. Nothing herein isto be construed as an admission that the inventors are not entitled toantedate such disclosure by virtue of prior invention or that they areotherwise part of the prior art.

SUMMARY OF THE INVENTION

In one aspect, the present invention is a method of selecting cells withenhanced secretion of a secretory product including the following steps:(a) providing a population of cells including cells in which increasedintracellular concentrations of calcium ions is correlated with theextracellular presence of a secretagogue; (b) exposing the population tothe secretagogue in concentration sufficient to result in secretion ofthe protein; (c) selecting from the population, those cells whichexhibit increased amounts of intracellular free calcium when exposed tothe secretagogue in step (b); and (d) culturing the selected cells.

In another aspect, the present invention is a method of cloning β cellsincluding the following steps: providing a population of β cells;proliferating the cells in the population on soft agar; selectingindividual clusters of the cells in the population to create subclones;dissociating the subclones; and proliferating the subclonal cells toproduce subclonal cell lines.

In still another aspect, the present invention is a method of producinga correctly regulated population of β cells including the followingsteps: (a) providing a population of correctly regulated β cells; and(b) selecting from the population, a group of cells which divide slowlyrelative to other cells in the population.

In still another aspect of the present invention, a method of providinga line of correctly regulated β cells including the following steps:providing a population of β cells; selecting from said population thecells which secrete approximately twice as much insulin when stimulatedby glucose at a first concentration at a point above about 10 mM as theydo when stimulated by glucose at a second concentration at a point inthe range of about 3 mM to about 9 mM.

In yet another aspect of the present invention, a line of correctlyregulated β cells is provided capable of secreting more than about 1300μUnits of insulin/45 minutes/50,000 cells.

In yet another aspect of the present invention, a line of β cells isprovided capable of maintaining insulin secretion levels of more thanabout 1300 μUnits insulin/45 minutes/50,000 cells for more than about 25passages in culture.

In yet another aspect of the present invention, a line of correctlyregulated β cells is provided capable of secreting more than about 20%of their insulin content in response to maximal levels of glucose.

In still a further aspect of the present invention, a collection ofcorrectly regulated β cells is provided containing a vital dye.

Other aspects of the invention are provided to accomplish the desiredgoal set forth above.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph illustrating the glucose responsiveness of a correctlyregulated idealized islet cell. The dotted lines shows optimum glucoseresponsiveness, while the solid black curves illustrate the outer rangesof glucose responsiveness in cells considered to be "correctlyregulated" in response to glucose.

FIGS. 2A through 2D are graphs illustrating the glucose responsivenessof prior art β-TC-6 cells at passages 17, 26, 31, and 33 respectively.

FIG. 3 is a graph of insulin released expressed as a percent of totalcellular insulin content at various levels of glucose stimulation, of aclonal cell subline according to the present invention.

FIGS. 4A-4D are graphs of the glucose responsiveness of four selectedclonal cell sublines according to the present invention.

FIGS. 5A and 5B are the dot plots of the flow cytometry data used in thecell sorting method of the present invention illustrating the increasednumber of cells having a fluorescent intensity labelled R1 at themaximal glucose level (FIG. 5B) versus the half-maximal glucose level(FIG. 5A).

FIG. 6 illustrates the relative insulin released at 16 mM glucose ascompared to 8 mM, by the parent cells, the subline produced by sortingaccording to the present invention ("Sβ-1"), and another subline ("T6")produced by sorting more than about 10 minutes after the cells wereexposed to glucose.

FIG. 7 is a graph illustrating the glucose responsiveness of cellsproduced according to the sorting method of the present invention atpassages 37 and 38.

FIG. 8 is a graph of the insulin secretion of perifused cells producedby sorting according to the present invention, taken over time withstepped increases in glucose level.

FIG. 9 is a graph of the insulin production of perifused adult mouseislet cells in primary tissue culture over time taken at steppedincreases in glucose level.

FIG. 10 is a graph of the average insulin production by perifusedporcine islet cells in primary tissue culture in response to high butconstant glucose stimulation over time for 22 separate islet isolationsand perifusions.

FIG. 11 is a graph of insulin released versus glucose concentration insubclone F7-1 at passage 38 with and without exposure to IBMX and othersecretagogues.

FIG. 12 is a graph of the amount of insulin released as a percent oftotal cellular insulin content in cells sorted according to the presentinvention at increasing levels of glucose, measured 12 passages aftersorting.

FIGS. 13A-13D show dot plots of cells to be sorted according to rate ofdivision using cell membrane markers according to the present invention.

DETAILED DESCRIPTION OF THE SPECIFIC EMBODIMENTS

Physiological characterization of 6 cells as they exist in the endocrinepancreas has led to a general understanding of the glucose response andinsulin secretory dynamics required to maintain normal blood glucoselevels. Studies on isolated islets in vitro have confirmed much of theunderstanding gleaned from in vivo studies, and additionally thetransplantation of isolated human pancreatic islets which had previouslybeen characterized in vitro allows the prediction of the insulinsecretory dynamics that would be required for a dividing cell in orderto be physiological or therapeutically useful. In general, then, usefulβ cells will release useful amounts of insulin in response to glucosewith similar regulation characteristics as healthy non-dividing isletcells in vitro. Glucose regulation similar to islets in vitro will bereferred to herein as "correct regulation." This characteristic responseto glucose appears as a sigmoidal curve on a graph of insulin secretionversus glucose concentration.

The dotted line in FIG. 1 shows an idealized plot of insulin release atvarious glucose concentrations. In some embodiments, the instantinvention provides for the preparation of cell lines which have glucoseresponse characteristics which fall within the area bounded by the twosolid lines. Such response characteristics are characterized herein as"correct regulation." In preferred embodiments, the cell lines havecharacteristics which are close to or the same as the dotted line. Thus,the cells of the instant invention exhibit maximal insulin release atabout 10 mM glucose or more. "Half-maximal level" as used hereafterrefers to a) the glucose level at which the cells secrete about half themaximum amount when the level of maximum insulin release is at 16 mMglucose or less, or b) the glucose level at which the cells secreteabout one-half the amount of insulin they secrete in 16 mM glucoseconcentrations when the maximum secretion level is at more than 16 mMglucose.

A correctly regulated cell line according to the present invention showsa half-maximal amount of insulin secretion at a glucose concentrationranging from about 3 mM to 10 mM, preferably about 4 mM to 9 mM, andoptimally about 5 to 6 mM. In a correctly regulated line, maximalinsulin secretion occurs at a glucose concentration of about 10 mM ormore, usually about 12 mM to about 20 mM, and optimally at about 16 mM.In addition, basal insulin release in correctly regulated cells isrepresented by the portion of the glucose response curve less than about1 mM glucose. The transition of the slope of the line in the basalregion (when glucose concentration<1 mM) to the slope at thehalf-maximal point is greater than 50% complete (change inslope>((half-maximal slope -basal slope))/2) at glucose concentrationsgreater than 1 mM, most preferably at concentrations greater than about2 mM and most preferably at concentrations greater than about 3 mM.Finally, in a "correctly regulated" cell population, the maximum amountof insulin secretion is about 4 or more times, preferably 6 or moretimes, most preferably 8 or more times that at the basal concentrationof glucose. By way of information, a 16 mM glucose concentration isequivalent to a 300 mg/dl glucose solution. This is a representativenormal in vivo high blood glucose concentration, although diabetics willsometimes have a higher blood glucose concentration of about 400 mg/dl.Where a diabetic's blood level rises above 400 mg/dl level, there issubstantial risk to the organism.

Prior cell lines (the RIN cell lines, the NIT cell lines, the HIT celllines, the β-TC cell lines, the INS cell lines, the MIN cell lines) asdiscussed earlier have generally failed to simulate normal islet cellsin one or more of the following respects: lower than desirable insulinsecretion levels, instability of secretion characteristics in culture,incorrect regulation of insulin, lack of reproducible means forrecreating the cell lines, and/or special culturing requirements. Todate, researchers have not been able to obtain insulin production atlevels above about 1125 μIU insulin/45 min/10⁵ cells for numerouspassages in culture in any cell line or population produced.

In one embodiment, the present invention is a repeatable method ofproducing a cell population or line which secretes a given protein,preferably insulin, at enhanced levels and/or in a correctly regulatedmanner, for enhanced numbers of passages in culture. The protein isproduced in response to a "secretagogue" (hereby defined as a compoundor composition in response to which the cells secrete the protein) whichis preferably glucose, and the cells are preferably β cells.

In one preferred embodiment, the method of producing cells with enhancedsecretion of a protein includes the following steps:

(a) providing a population of cells including cells in which increasedintracellular concentrations of calcium ions is correlated with theextracellular presence of a secretagogue;

(b) exposing the cells to a secretagogue in concentration sufficient toresult in secretion of the protein;

(c) selecting from the cells those which exhibit increased amounts ofintracellular free calcium when exposed to the secretogogue in step (b);

(d) culturing the selected cells.

The population of cells provided in step (a) is a population ofsecretory cells in which increases in intracellular calcium arecorrelated with exposure to a secretagogue, i.e., in which increases inintracellular calcium are implicated in secretion of a desired protein,in response to application of a secretagogue. Examples of such cells areneurosecretory- or hormone-producing cells (which produceneurotransmitters or proteins). Examples of hormone producing cells areβ cells and pituitary-derived cells. Secretory products for β cellsinclude GABA (gamma-aminobutyric acid) as well as insulin.Adrenocorticotrophic hormone (ACTH) is secreted by the AT T20 cell line.

Examples of neurosecretory cells are adrenal chromaffin cells, neurons,glia, and the like. Adrenal chromaffin cells secrete opioid peptidessuch as enkephalin in response to nicotine or acetylcholine and alsosecrete epinephrine and norepinephrine while certain neurons secreteglutamate. Cells derived from adrenal medulla cells, as well asembryonic ventral mesencephalic tissues, and cells derived from theneuroblastic cells secrete dopamine. Examples of neurosecretory celllines are GT-1 which produces gonadotropin releasing hormone (GNDH) andPC-12 which secretes dopamine. In the preferred embodiment, the cellsare β cells and the secretory product is insulin. The secretagogue canbe any of a number of compounds such as L-leucine, α-ketoisocaproicacid, d-glyceraldehyde, arginine, glucagon, gastric inhibitory peptide,carbamylcholine, and potassium (at high levels). The preferredsecretagogue is glucose.

The starting population of β cells used preferably show measurablelevels of insulin production, i.e., greater than 100 μIU/45 min/10⁵cells, preferably greater than 1000 μIU/45 min/10⁵ cells, mostpreferably greater than 4000 μIU/45 min/10⁵ cells, such as the β-TC-6cell line. It is also preferable that the starting population of cellsfor the present invention show correct regulation at some passage. Itshould be noted in this regard that the cells selected need not beselected at a passage in which they show correct regulation. In thepreferred embodiment, the cells were sorted at passage 21, whileregulation of the parent line deteriorated sometime between passage 17and 26. (The word "passage" as used herein refers to the transfer ofcells in culture from one media to another after reaching agrowth-limiting concentration in the first media.)

FIGS. 2A through 2D illustrate insulin secretion of the β-TC-6 cell lineused as a parent cell population in the preferred embodiment of thepresent invention. At passage 17 (see FIGS. 2A and 2B), it can be seenthat the β-TC-6 cells show some insulin regulation and a sigmoidal curvein response to increased levels of glucose although the relative amountsof insulin secreted indicate that the cell population is not "correctlyregulated" at passage 17. At passage 26 (see FIG. 2B), regulation hasdeteriorated, the sigmoidal curve disappearing, and at passages 31 and33 (see FIGS. 2C and 2D) regulation has completely disappeared, lowlevels of insulin being secreted at all glucose levels. Thus, overnumerous passages, control of insulin secretion in β-TC-6 cells hassignificantly degraded.

However, it is not essential that the present invention utilize anexisting cell line or population as the parent population. Instead, abase population of cells can be produced for use as the parentpopulation using known methods. For example, Efrat, S., Linde, S.,Kofod, H., Specter, D., Delannoy, M., Grant, S., Hanahan, D., andBaekkeskov, S., "Beta-cell lines derived from transgenic mice expressinga hybrid insulin gene-oncogene", in Proc. Natl. Acad. Sci. U.S.A. (1988)85:9037-9041, which is incorporated herein as though fully set forth, onpage 9037 teach a method for producing β-TC cells which can be used asthe parent population for the methods of the present invention.Likewise, Radvanyl, et al., (1993) Molecular and Cellular Biology. Vol7., No. 7, pp. 4223-4232, which is hereby incorporated by reference asthough fully set forth discloses a method for producing cell lines fromhyperplastic β cell populations.

In the preferred method, the cells are exposed in step (b) to levels ofthe secretagogue which are known to stimulate protein secretion in thecells of interest.

Preferably, the above method includes a further step (e) of exposing thepopulation to a calcium-activated labelling agent. The preferredlabelling agents are vital (non-toxic) dyes which complex with Ca⁺² andfluoresce at certain wavelengths and thus can be utilized forfluorescence-activated cell sorting. An example is Fluo-3-acetoxymethylester ("Fluo-3") or other dyes such as Indo-1 acetoxymethyl ester("Indo-1") all made by Molecular Probes, Eugene, Oreg. The dyes showsensitivity to calcium ions so that increases in calcium content at thelevels of interest are visible. In one aspect, the present inventioncomprises correctly regulated cells, such as those of the presentinvention, including vital dyes.

In the preferred method of the present invention, the labelled cells areexposed to a lower and a higher secretagogue concentration. Preferablythe lower concentration is at a half-maximal level and the higherconcentration is at a maximal level. Cell labelling is then preferablyassessed.

In the preferred embodiment, the sorting is accomplished byfluorescence-activated cell sorting. The preferred method isfluorescence-activated cell sorting using the calcium-activatedlabelling agents mentioned above. After flow cytometry at thehalf-maximal and maximal glucose levels, a fluorescent intensity isdefined to determine which group of cells will be selected in thesorting method of the present invention. The fluorescent intensity isselected such that the number of labelled cells having more fluorescencethan the selected amount in the higher secretagogue concentration isgreater than the number having more fluorescence than the selectedamount in the lower secretagogue concentration. Most preferably, thefluorescent intensity is selected to define a group of cells in thehigher glucose concentration containing about 50% more cells than thatfound at the lower concentration. More preferably the selected group inthe higher concentration contains about twice as many cells as in thelower concentration. Most preferably, the number of cells will be about20 times greater in the higher compared to the lower concentrationpopulation.

Referring to FIG. 6A, a dot plot is shown of the flow cytometry of anembodiment of the present invention at a half- maximal glucoseconcentration, while FIG. 6B shows the dot plot at the maximal glucoseconcentration. The group of cells falling within the area labelled R1 isselected. 900 cells appear in R1 at the maximal glucose concentrationwhile only 552 appear at the half-maximal glucose concentration.

The same technique can be used to select insulin-selecting secretingcells correctly regulated for other secretagogues such as cholinergicagonists, amino acids, and peptides such as glucagon.

Another aspect of the invention is the cell populations or cell linesproduced according to the above-described method. Insulin production incells of the present invention is preferably more than about 900 μIU("International Units")/45 min/50,000 cells plated, more preferably morethan about 1300 μIU/45 min/50,000 plated cells, most preferably about2000 μIU/45 min/50,000 plated cells. (It should be noted that cellsproduced by the above method were plated at 50,000 cells; and testedafter dilution and proliferation. A sample is thus expected to containabout 10⁵ cells, due to the amount of proliferation so that actualpreferred insulin level is about 2,000 μIU/45 min/10⁵ cells, forexample.) In another embodiment, the amount of insulin released from thecells is preferably about 20%, more preferably about 20%, or mostpreferably about 60 to 70% or more of the total insulin content as themaximum level of insulin secretion. The cells are also preferablycorrectly regulated.

The preferred cell populations or lines of the present invention havebeen found to maintain their insulin-producing characteristics and/ortheir correct regulation more than about 5, more preferably more thanabout 23, most preferably more than about 30 passages in culture aftertheir preparation.

In another aspect, the invention is another method of producing a lineof β cells which are high insulin producers and/or are correctlyregulated over numerous passages in culture. The cell line is created byproviding a population of β cells, growing the cell population or cellline in soft agar, dissociating the cells, preferably using trypsin,although other dissociating agents such as collagenase, pancreatin, orany enzyme generally useful for cell dissociation for tissue culturescan be used, cloning individual cell clusters, and selecting those withdesired insulin-responsiveness. "Cloning" as used herein refers to theprocess of culturing individual cells taken from a group of cells, toform a line of identical cells. The subclonal cell lines produced havethe characteristics described above and are another aspect of thepresent invention. In the preferred embodiment, approximately five ofthe 40 subclonal lines were selected to form the subclonal cell lines ofthe present invention.

The soft agar used has been found to allow the cells to grow inthree-dimensional rather than flat clusters, and thus apparently toremain more differentiated during growth. Soft agar as used herein meansagar which is sufficiently viscous to allow such three-dimensionalgrowth and contains about 0.1 to about 1% of actual agar, mostpreferably about 0.3% agar. Soft agar is made by dissolving anappropriate amount of agar in water. Alternatives to soft agar may bealginate and agarose. In order to promote growth, a desirable agar willcontain growth factors such as laminen, type 4 collagen and basic F6F ortheir equivalents. The preferred material is Matrigel™ produced byCollaborative Research which contains such growth factors.

During cloning, the cells are preferably fed with standard media such asDulbecco's Modified Eagle's Medium ("DMEM") having about 5 to about 30%,preferably 10% by volume conditioned media from other β cells, such asβ-TC-3 cells. The conditioned media is believed to help maintain thedifferentiation of the cells in culture. After growing in this media,the cells are harvested and tested for insulin secretion. Clones arethen selected for insulin production, subclones having high insulinproduction being desirable; in the preferred embodiment, subclonesshowing about twice as much insulin production at the maximal glucoselevel as at the half-maximal level of glucose solutions are selected.Data showing results of such screening for the parent line, and cells ofthe present invention is graphed in FIG. 7.

In the preferred embodiment, a β cell line is produced with correctlyregulated insulin production over numerous passages in culture or invivo and the cell line or population itself is another aspect of theinvention. The cell line produced shows the insulin secretion levelsdiscussed above.

Another embodiment of the invention is a method of producing apopulation, preferably a cell line, of correctly regulated β cellshaving high insulin secretion, preferably a cell line of such cells. Inthis embodiment, correctly regulated cells are selected according totheir rate of division. Generally, the method includes selecting acorrectly regulated starting population such as that produced accordingto the sorting or cloning methods of the present invention. The methodincludes the step of selecting these cells, if they are slowly dividing;otherwise dividing them into a rapidly dividing and a slowly dividingpopulation, and selecting slowly dividing subpopulation producing moreinsulin than the rapidly dividing subpopulation.

In the preferred embodiment, selecting is accomplished usingfluorescence-activated cell sorting and a cell membrane marker. The cellmembrane marker is placed in solution with the cells to label the cellmembrane in a fashion which is visible in the cell sorter. Flowcytometry data on the labelled cells is obtained immediately aftermarking. A portion of the cells showing high marking (Gate R4 on FIG.14A) is selected. The selected cells are then allowed to proliferate. Inthe preferred embodiment, any vital cell membrane markers can be used ina fashion known in the art but the preferred cell membrane marker isPKH26-GL produced by Molecular Probes, Eugene, Oregon. Cells are usuallyexposed to the marker in concentrations which render them visible underfluorescence.

Using the sorter, the cells (preferably those selected as R4), afterproliferation, are divided into two populations according to the amountof labelling, the highly marked population being the one that isselected. In the preferred embodiment, the cells selected show ahalf-life decay of fluorescence intensity which is greater than about30%, more preferably greater than about 100% of the mean doubling timeof the population. FIG. 14 is a dot plot of the results of flowcytometry of cells produced according to the above invention. In FIG.13A, gate R4 shows the high fluorescence of most of the cells of thepresent invention immediately after labelling while gates R1 through R3illustrate lower levels of fluorescence. After a few days of culturing(FIG. 13B), fewer cells show the high fluorescence level of gate R4.After another week in culture (FIG. 13C), the bulk of the cells showfluorescence clustering around gate R2 and R3; only a few cells show thehigh fluorescence of gate R4. After 2 weeks in culture, only a few cellsshow the high fluorescence of gate R4 (FIG. 13D) and they are selected.

In another embodiment, the present invention is a method of producinghigh insulin producing and/or correctly regulated, stable cell linesfrom a parent population of β cells by selecting the cells in whichabout twice as much insulin is secreted in about a 10 mM or more glucosesolution as in about 3 mM to about 9 mM. Preferably, the first glucoseconcentration is about 12 mM to about 20 mM and the second concentrationas about 5 mM to 9 mM. Most preferably, the first glucose concentrationis about 16 mM and the second concentration is about 5-6 mM, the firstconcentration usually being at the maximal level and the second being atthe half-maximal level. The cells are preferably selected according tothe above-mentioned sorting or cloning processes.

In another embodiment of the invention, cells produced according to thepresent invention are enclosed in biocompatible insulin-permeablemembranes for implantation. The membranes are preferably hollow fibermembranes or flat sheets which are considered particularly appropriatefor implantation into humans. Preferred materials, the construction ofthe devices and, and the manner of enclosing the cells within them aredisclosed fully in U.S. patent application Ser. Nos. 08/082,407, filedJun. 23, 1993 and PCT/US92/03327, filed Apr. 22, 1992, and incorporatedherein as though fully set forth. Generally, the cells of the presentinvention will be loaded into the device in the presence of solublealginate which can subsequently by polymerized by placement of thedevice into a calcium chloride containing medium.

In another embodiment, the invention is a method of treating diabetes byimplanting the encapsulated or enclosed cells described above. Numbersof cells and encapsulating devices to be implanted are preferablydetermined by traditional methods for determining insulin dosage in adiabetic patient. Large scale devices can be prepared as described inU.S. patent application PCT/US92/03327 above, and implanted intoperitoneal or subcutaneous locations. Sufficient cells will be placedwithin the device so that patients will achieve normolycemina over atwenty-four hour period. The exact size of the device to deliver theproper dose will be determined empirically, but comparison of theinsulin output of small devices used to cure diabetes in rodents (seeU.S. patent application Ser. No. PCT/US92/03327, supra) will allowappropriate scaling for the body weight of a patient.

Implanting is accomplished by surgical methods known in the art.

EXAMPLES Example 1: Selection of Subclones of β-TC-6

β-TC-6 cells were obtained and cultured. Glucose responsiveness wastested at passages 17, 26, 31, and 33 by a modification of the method ofEfrat, et al. (1993) as shown in FIGS. 2A through 2D. Briefly, cellswere plated in 24 well falcon tissue culture plates at a density of50,000 cells/well. The media was removed and cultures were rinsed 2-3times with either Krebs solution, or Modified Eagle's Medium (MEM)containing 10% horse serum and 25 mM Hepes. Then, 1.5 ml of MEM with 10%HS and 0.5 mM IBMX was added to each well and cells were returned to theincubator. After 30 minutes, 0.5 ml of media was removed from each well(preincubation samples), and the glucose concentration of the remainingmedia in the wells was adjusted using a 30 mg/ml glucose stock and cellswere returned to the incubator for an additional 45 minutes. At thatpoint, media was harvested and assayed for insulin content using a radioimmunoassay. The insulin released during the 45 minute incubation periodwas then calculated by subtracting the preincubation values from the 45minute sample. Generally, 4 wells were used per glucose concentration.While passage 17 showed some regulated insulin production, regulationwas not "correct" as discussed above and passage 26 showed deterioratinginsulin production. Passages 31 and 33 showed poor regulation, withlittle or no increased insulin production upon exposure to increasedlevels of glucose.

About 60,000 passage 18 cells were suspended in 9 mls pre-warmed"complete" media at 37° C. (Complete media was Dulbecco's ModifiedEagle's Medium ("DMEM"), Gibco No. 320-1965 with 15% horse serum byvolume (all references to solutions and percentages herein are byvolume, unless indicated otherwise) 2.5% fetal bovine serum (Gibco) and10% conditioned media from β-TC-3 cells.) A half mil of Matrigel™(Collaborative Research) was added to the solution. Soft agar was madeby dissolving an appropriate amount of agar (made by Batco) in water.The cell suspension previously made was then mixed with the agarsolution. 1.5 mls of the agar/cell solution was then placed in each wellof a well plate.

Cells were allowed to grow in the agar for three weeks, being fed with10% conditioned media twice a week. Individual cell clusters wereharvested by pipet, and each cell cluster was placed in a well of a wellplate to create a subclone, 40 subclones being created. Clusters werebroken apart and fed three times a week with the above media, allowed toproliferate, and then split to expand the number of cultures. Eachsubclone was tested as a single well in a serial glucose response assay.Wells containing the cells of interest were rinsed several times withglucose-free Krebs or MEM containing 10% horse serum and 20 mM HEPES. 1ml of the same medium was then added to the cultures for 30 minutes. Atthat time, 0.5 mls of the media was removed (preincubation) and 0.5 mlsof fresh medium containing 6.4 mM glucose was added to the well to givea final glucose concentration of 3.2 mM. Cells were then incubated for30 minutes to 1 hour in the incubator at which time 0.5 mls was sampledand 0.5 ml of fresh media was added back containing enough glucose togive a final concentration of 8 mM glucose. The incubation period wasrepeated and a third sampling was performed at which time the glucoseconcentration was adjusted to 16 mM with another 0.5 ml of glucosecontaining medium. All samples were frozen and subsequently assayed forinsulin content. Amounts of insulin released during a given incubationwere calculated by subtracting the residual insulin levels from thenewly released amounts.

Individual wells containing subclones showing about twice as muchinsulin secretion in 16 mM glucose solutions as 8 mM glucose solutionswere selected in order to develop the cell lines of the presentinvention. Once the cloned line has been expanded to the point thatappreciable levels of insulin can be detected (e.g., 5,000-50,000 cellsor 14-17 doublings) a single culture can be assayed using a serialglucose challenge where, following a rinse procedure, the same cultureis exposed to stepwise increases in glucose concentration and a sampleof the media is removed before each increment in glucose concentration,and insulin release values calculated. Useful concentrations for suchassays include a low value such as 3.2 mM glucose or less, a value ofglucose around the expected half-maximal response such as 8 mM and amaximum stimulatory concentration such as 16 or 20 mM. In such serialassays where a relatively small number of cells are being assayed,sometimes artificially elevated insulin levels are encountered inresponse to the first (i.e., lowest) glucose concentration. Therefore,generally it is best to consider only the insulin output at theanticipated half-max and maximal values. Those populations with greaterthan a two-fold difference between an 8 mM and 16 mM glucoseconcentration can be considered as likely to be correctly regulated andthe cultures can be further expanded for determination of completeglucose response curves.

The selected subclones were then cultured in the complete mediamentioned above. At passage 32 (13 passages after cloning), they weretested for glucose-responsiveness using the assay method mentionedabove. The cells were still correctly regulated, and a sigmoidal insulinresponse curve was obtained. FIGS. 4A and 4B illustrate the insulinresponse curve for four of the subclones at passage 32 in the culture.Maximum insulin secretion ranged from about 4500 to about 8200 μIUinsulin/45 min/50,000 cells plated. FIG. 3 illustrates the percent ofintracellular insulin released by subclone F7-1, one of the selectedsubclones.

Example 2: Selection and Cloning of Cell Lines by Calcium ActivatedFluorescent Cell Sorting

Preparation of Cells to be Stained

β-TC-6 cells at passage 21 were trypsinized and resuspended in cellculture media, taking care to make a single cell suspension. Cells werecounted and approximately 1-2×10⁶ cells were placed in each tube. Onetube was retained as a background control and was unstained. Cells werewashed twice with Hanks' Balanced Salt Solution (HBSS) with 1% serum.

Preparation of Dye Stock

Immediately prior to staining the cells, the stocks and workingsolutions were made. To a 50 microgram aliquot of Fluo-3 acetoxymethylester™ made by Molecular Probes (F-1242, stored desiccated in freezer)were added, in order:

35 micro liters of Pluronic F-127TN stock (Basfyandott) (stored inscintillation viaswrappedsin foil) Pluronic is a non-ionic, highmolecular weight surfactant polyol useful for helping solubilizewater-insoluble dyes.

113 microliters fetal bovine serum (FBS) The latter was pipetted up anddown to reconstitute the dye.

The dye was orange in color when fully resuspended. The stock waswrapped in foil and placed in the freezer.

Preparation of a Working Solution

To make a 1.2 micromolar working solution, 30 microliters of the dyestock was added to 10 mls of HBSS with 1% serum. The vessel was wrappedwith foil and held at room temperature.

Staining of Cells

1 ml of the 1.2 micromolar working solution of Fluo-3 was added to eachpellet of cells (except for the background cells). The tubes were shakenlightly to disperse the dye. The tubes were wrapped in foil, and allowedto sit at room temperature for 30-45 minutes. The cells were washed withHBSS with 1% serum at 1000 rpm for 5 minutes. They were then washed inthe testing medium (Krebs or Modified Eagle's Medium (MEM), with 1%serum), and were rewrapped in foil and held for at least 39 minutes atroom temperature.

FacScan

Using a Becton Dickenson FACSort cell sorter, the background tubementioned above was used to set side scatter (SSC) and fluorescenceintensity (FSC) parameters so that all unstained cells of the backgroundcontrol were visible in the SSC/FSC dot plot. The FL-1 parameters wereset so that cells showed a fairly tight vertical distribution along theSSC axis. A first cell sample of approximately 10,000 labelled cells wasstimulated in a 100 mg/dl glucose solution by adding an appropriatealiquot of glucose from a 15-30 mg/ml glucose solution. The tube wasshaken lightly and the sample was immediately placed on the sample portto acquire flow cytometry data on the cells. The "Begin" switch wasactivated, and a scan was made of the cells, the dot plot of which isshown in FIG. 5A. The sample was similarly prepared at 300 mg/dl glucoseand flow cytometry data similarly obtained for it. The dot plot is shownin FIG. 5B. It was noted that the greatest increase in cell number atthe second concentration occurred at a fluorescent intensity justgreater than about 20 on the X-axis, so the area labelled R1 wasselected for sorting. While the cells could be scanned (or sorted) inthe first 9 minutes or more after the glucose was added, it waspreferred to scan them in the first 5, or more preferably the first 3minutes. Cells sorted more than 10 minutes after labelling with glucoseshowed poorer insulin production and are labelled T6 on FIG. 6.

FACSort

Parameters were set so that all cells in R1 are selected. The abovecells at about 300 mg/dl were then put into the sample port and acollection tube placed in the collection port, the "begin" switchactivated, and the cells were sorted from the group. Sorting wasaccomplished in the time frames set forth above for the SP-1 cell lineshown in FIG. 6; the T6 cell line was sorted after the cells remained inglucose more than 10 minutes.

Stability of Cell Line

The SP-1 cell line produced above was maintained in culture for 37-38passages. At that time, it was tested for glucose responsiveness usingthe same assay mentioned above; it showed correct insulin regulation anda capacity to produce 4000-5000 μIU insulin/45 min/50,000 cells whenstimulated by 16 mM glucose. A graph of these results is shown in FIG.7.

Insulin Regulation in Sorted Cells:

Sβ-1 cells at passage 3 were characterized in a stepped perifusion studyafter 24 hours in culture medium including IBMX. The results shown inFIG. 8 were normalized for DNA since the cells contain more DNA per cellthan the islet cells used for comparison (see later discussion). Thecells show almost 25 μIU insulin release per mil per μg DNA.

Adult mouse islet cells were similarly tested and also showed a peak at16.7 mM glucose concentration. The results are shown in FIG. 9. Porcineislet cells were also tested over time but at a constant in glucoselevel as shown in FIG. 10. Porcine islet cells are of particularinterest as a comparison because they are believed to closely mimicnormal human cells and are therefore frequently used as a model forhuman islets. The porcine cells when exposed to insulin at high levelsfor about 2 hours show a brief peak at about 30 to 35 minutes andanother peak at about 90 to 100 minutes. During the period from about 40minutes to 90 minutes, they were shown to release a constant amount ofinsulin, specifically, about 60 μIU insulin/ml/mg of DNA.

Example 3: Selection of Slowly Dividing Cell Lines usingFluorescent-Activated Sorting and "Cell Link" Labeling

Cells of the Sβ-1 subline produced by the fluorescence-activated sortingdescribed above were trypsinized and washed with calcium- andmagnesium-free Hanks' Balanced Salt Solution and centrifuged at 400×gfor 5 minutes. Cells were then resuspended in 1 mil of (the same)diluent and mixed with 2×solution (4×10⁻⁶ MM) of the cell membranemarker PKH26-GL and incubated for 2 to 5 minutes. Two mls of horse serumwere added to quench the staining. After 1 minute in serum, 4 mls ofcomplete Dulbecco's Modified Eagle's Medium (DMEM) were added and thesolution centrifuged for 10 minutes at 400×g. The supernatant wasaspirated and the wash procedure repeated a total of three times. Thecells were returned to culture. The stained cultures were measured byflow cytometry on days 1, 5, 12 and 14 for remaining fluorescence (sidescatter) and were then sorted. The data is presented in FIGS. 13A and13B which illustrates side scatter on the vertical axis versusfluorescence intensity on the horizontal axis. A small population (about1%) of highly fluorescent cells (gate=R4) were present after 14 days(FIG. 13D) in culture indicating the presence of very slowly dividingcell in the cultures.

To select for cells and produce a cell line showing high insulinproduction, a population of β cells, preferably an already sortedpopulation such as the Sβ-1 line, is labeled as above, and the narrowband (gate=R4) of highly fluorescent cells is collected. These cells arereturned to culture; after one week or more, the cells are harvested,the distribution of fluorescent intensity is determined and the mosthighly fluorescent (gate=R4) are sorted and placed in culture. Thispopulation is then assayed for insulin output and is expected to producehigher insulin levels while remaining correctly regulated.

Example 4: Method of Encapsulation of β-Cells

β-cell line cultures were prepared according to Example 1. After 10-30passages in culture, the cells are harvested using a sterile pipet.Cells are washed in CMRL 1066™ (Gibco) and resuspended in CMRL 1066(Gibco) to a concentration of 25 million/ml. A 2% solution of sodiumalginate is prepared under sterile conditions. The cells are diluted 1:1with the alginate solution, for a final concentration of 1% alginate inthe islet suspension. The cells are hand-loaded into a PAN/PVCpermselective hollow fiber membrane according to the method of Dionne inU.S. patent application Ser. No. PCT/US92/03327, filed on Apr. 2, 1992,which is incorporated herein as though fully set forth. The fiberdevices are sealed and placed in a 1% calcium chloride solution to crosslink the alginate. The fibers are placed in CMRL 1066 overnight. Thefibers are tested for glucose responsivity by static and perifusionchallenge with 0.1, 3.3, 8.3, 16.7 mM glucose.

The encapsulation procedure was also performed for parent β-TC-6 cellsat about passages 15 to 17 (during which they are known to be correctlyregulated). Correct regulation and a standard sigmoidal curve forglucose responsiveness was found in two of the three batches tested.Also, maximum insulin release at high glucose levels was found.

Example 5: Implantation of Encapsulated Cells into the Mouse

Based on the information obtained above relating to insulin secretion,and the insulin requirements of the mouse, it was determined thatapproximately 7 million cells need to be implanted in a mouse tomaintain the mouse normoglycemic. The volume of this number of cells isabout 10 μl. At a final cell density (after proliferation) of about7-10% by volume, it was determined that approximately seven hollowfibers of 1000 μm i.d. and about 2.5 cm long are required to beimplanted.

A correctly glucose responsive β-cell line was prepared according toExample 1 or 2. Cells are encapsulated according to Example 3. Devicesare implanted intraperitoneally into streptozotocin-induced diabeticmice. Plasma glucose levels are monitored daily. After 60 days, thedevices are removed and the mice are checked for a return tohyperglycemia. The recovered devices are assessed for the ability torelease insulin in response to glucose perifusion.

Example 6: Implantation of Encapsulated β-cells in Human subject

Based on insulation rates of the cells used and the amount of insulinneeded to maintain normoglycemia over a 24 hour period in a humanpatient, it was determined that about 600-700 million cells need to beimplanted to maintain a human normoglycemic. To produce this number ofcells once proliferation has occurred, approximately 17 million cells ofthe above need to be implanted. Thus, 10 6 cm diameter flat sheets arerequired, containing, after proliferation, 65 million cells each, torelease 40-60 IU insulin/day. 70 μl of an alginate cell slurrycontaining 25 million cells/ml is to be evenly spread throughout eachdevice.

A β-cell suspension in alginate is prepared according to Example 3, at adensity of 25 million cells/ml. Seventy microliters are loaded into eachof 10 flat sheet devices, each having a surface area of 70 cm²(including both sides) and are sealed according to the method disclosedin U.S. patent application Ser. No. 08/082,407, filed Jun. 23, 1993,incorporated herein as though fully set forth.

The devices are implanted in peritoneal cavity, preferably in and aroundlobes of the liver. After implantation, intensive insulin treatment (2-4shots of insulin and 10-15 glucose checks per day) is maintained for 2weeks. Exogenous insulin is slowly tapered off as cell number increasesand more insulin is released from the devices. Patients are removed frominsulin and returned to normal glucose monitoring 1-2 months postimplantation.

Example 7: Effect of Other Agents on Insulin Secretion

All insulin assays performed on the cells reported herein were done inthe presence of 0.5 mM IBMX. IBMX is a potent enhancer of insulinsecretion, which does not change the regulatory characteristics of p TCcells. Efrat et al, 1993 has shown that IBMX potentiates insulin releaseup to approximately 3-fold. However, if cells are to be used in vivowhere they will not be exposed to IBMX, there are a number of othersecretagogues and secretion enhancers which circulate systemically.These include glucagon, gastric inhibitory peptide, and the amino acidsleucine and arginine. Therefore, it was also of interest to see if acocktail of these natural agents when presented to the P cells of thepresent invention at physiologically relevant concentrations wouldenhance insulin secretion in vitro.

F7-1 cells were plated at a concentration of 50,000 cells/ml accordingto standard insulin assay conditions and were assayed as described inExample 1 except that in some cases the preincubation buffer containedeither 0.5 mM IBMX or a secretagogue cocktail which consisted of:

0.013 mg/ml leucine

0.014 mg/ml arginine

0.016 mg/ml phenylalanine

0.001 μg/ml growth hormone

0.3 μg/ml glucagon

2 ng/ml gastric inhibitory peptide

At 16 mM glucose, the secretagogue cocktail was approximately half aspotent as IBMX alone, indicating that these cells are likely to haveenhanced insulin secretion in vivo as compared to in vitro Additionally,the enhancement of insulin secretion by IBMX at 16 mM glucose was lessthan two-fold.

It will be understood that the above discussion is intended by way ofdescription and not by way of limitation and that many other embodimentswill be apparent to those of skill in the art and will fall within thescope of the invention as defined by the appended claims.

What is claimed is:
 1. A line of β cells capable of maintaining insulinsecretion levels of more than about 1300 μUnits insulin/45minutes/50,000 cells for more than about 25 passages in culture.
 2. Aline of cells according to claim 1 and wherein the cells are correctlyregulated.
 3. A line of cells according to claim 1 and wherein the cellsare capable of secreting more than about 2500 μUnits insulin/45minutes/50,000 cells for said 25 passages.